ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells Maidina Tuohetahuntila,* Martijn R. Molenaar,* Bart Spee,† Jos F. Brouwers,* Martin Houweling,* Arie B. Vaandrager,* and J. Bernd Helms1,* Departments of Biochemistry and Cell Biology* and Clinical Sciences of Companion Animals,† Faculty of Veterinary Medicine and Institute of Biomembranes, Utrecht University, 3584 CM Utrecht, The Netherlands

Abstract  Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, -smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.—Tuohetahuntila, M., M. R. Molenaar, B. Spee, J. F. Brouwers, M. Houweling, A. B. Vaandrager, and J. B. Helms. ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells. J. Lipid Res. 2016. 57: 1162–1174. Supplementary key words  vitamin A • lipase • lipolysis and fatty acid metabolism • lipid droplets • lipidomics • heavy isotope labeling • triacylglycerol pools • retinyl esters • adipose triglyceride lipase • diacylglycerol acyltransferase 1

Hepatic stellate cells (HSCs) are the main vitamin A (retinol)-storing cells of the body (1, 2). In a healthy liver, HSCs store vitamin A in the form of retinyl esters (REs) in large lipid droplets (LDs), together with triacylglycerols (TAGs) and cholesteryl esters (CEs). HSCs are located in the space of Disse, between the sinusoidal endothelial cells This work was supported by the Seventh Framework Programme of the European Union-funded “LipidomicNet” project (proposal number 202272). Manuscript received 8 January 2016 and in revised form 26 April 2016. Published, JLR Papers in Press, May 14, 2016 DOI 10.1194/jlr.M066415

and the hepatocytes. Upon liver injury, quiescent HSCs can transdifferentiate into an activated myofibroblastic phenotype (1). Activated macrophages, in concert with the HSCs, may initiate this transition by secreting cytokines, such as transforming growth factor  (TGF-), which stimulate the synthesis of matrix proteins and the release of retinoids by HSCs (1, 3). The loss of retinoids is associated with a gradual disappearance of the LDs inside the HSCs. We previously reported that LD degradation in activated rat HSCs occurs in two phases (4). Upon activation of the HSCs, the LDs reduce in size, but increase in number, during the first 7 days in culture before they disappear in a later phase. Raman and lipidomic studies showed that in the initial phase of HSC activation, the REs disappear rapidly, whereas the TAG content is transiently increased (4). Interestingly, this increase in TAGs in rat HSCs is predominantly caused by a large and specific increase in PUFA-containing TAG species during the first 7 days in culture, mediated by an increase in expression of the PUFA-specific FA, long-chain acyl-CoA synthase (ACSL)4, and a decrease in expression of the other ASCLs, especially ASCL1 (5). So far, the molecular mechanisms and identity of the enzymes involved in the observed increase in LD number and their subsequent breakdown during HSC activation are not well understood. An increase in number can be accomplished by the de novo synthesis of new LDs (6) or fission of existing large LDs (7). Breakdown of LDs is best characterized in adipose cells, in which key roles are assigned to adipose triglyceride lipase [ATGL, also known as patatin-like phospholipase domain containing (PNPLA)2], its coactivator, CGI-58, and hormonesensitive lipase (HSL) (8). The first two proteins are known

Abbreviations:  ACSL, long-chain acyl-CoA synthetase; APCI, atmospheric pressure chemical ionization; ATGL, adipose triglyceride lipase; CE, cholesteryl ester; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; HSC, hepatic stellate cell; HSL, hormone-sensitive lipase; LAL/Lipa, lysosomal acidic lipase; LD, lipid droplet; PF, paraformaldehyde; PNPLA, patatin-like phospholipase domain containing; RE, retinyl ester; -SMA, -smooth muscle actin; TAG, triacylglycerol. 1  To whom correspondence should be addressed.   e-mail: [email protected] The online version of this article (available at http://www.jlr.org) contains a supplement. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

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to have a more general function, as deficiencies in either one lead to neutral lipid storage diseases (9). Both ATGL and CGI-58 were present on LDs in the HSC line, HSC-T6 (10), and rat HSCs were shown to express ATGL, although it was downregulated upon activation (11). In mouse HSCs, lipid breakdown was shown to be partially mediated by a lipophagic pathway, as inhibition of autophagy increased the amount of LDs (12, 13). Because inhibition of autophagy was shown to impair HSC activation in mice and this effect could be partially reversed by addition of exogenous FAs, it was suggested that LD breakdown is required to fulfill the energy demands of HSCs during activation (13). On the other hand, HSC activation was shown to be relatively undisturbed in the absence of REs and LDs in lecithin:retinol acyltransferase (LRAT) knockout mice (14). In this study, we addressed the question of whether a change in lipid metabolism is causally related to the activation process in rat and mouse HSCs. We identified enzymes involved in LD formation and breakdown in HSCs in vitro and studied the effect of inhibition of these enzymes on HSC activation.

MATERIALS AND METHODS Reagents D4-palmitate, D8-arachidonate, and Atglistatin were purchased from Cayman Chemical (Ann Arbor, MI). DMEM, FBS, and penicillin/streptomycin were obtained from Gibco (Paisley, UK). BSA fraction V was obtained from PAA (Pasching, Austria). T863 and collagenase (Clostridium histolyticum type I) was obtained from Sigma-Aldrich (St. Louis, MO), and saponin from Riedel-de Haën (Seelze, Germany). The mouse monoclonal antibody against -smooth muscle actin (-SMA) was from Thermo Scientific (Waltham, MA). LD staining dye, LD540, was kindly donated by Dr. C. Thiele, Bonn, Germany. Hoechst 33342 was obtained from Molecular Probes (Paisley, UK), paraformaldehyde (PF) (8%) was obtained from Electron Microscopy Sciences (Hatfield, PA). Fluor­ Save was obtained from Calbiochem (Billerica, MA), all HPLC-MS solvents were from Biosolve (Valkenswaard, The Netherlands) with the exception of chloroform (Carl Roth, Karlsruhe, Germany) and were of HPLC grade. Silica-G (0.063–0.200 mm) was purchased from Merck (Darmstadt, Germany).

Animals We used 10- to 12-week-old male and female Atgl+/+ (WT) and Atgl/ mice, generated by crosses of Atgl+/ C57BL/6J mice (15) and paired on sex and age from the same nest and adult male Wistar rats (300–400 g). Procedures of mouse and rat care and handling were in accordance with governmental and international guidelines on animal experimentation, and were approved by the Animal Experimentation Committee (Dierexperimentencommissie) of Utrecht University (Dierexperimentencommissie numbers: 2010.III.09.110, 2012.III.10.100, and 2013.III.09.065).

HSC isolation and in vitro primary cell culture Stellate cells were isolated from livers of mice and rats by collagenase digestion followed by differential centrifugation (16) and cultured, as described previously (5), in DMEM supplemented

with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 4 l/ml Fungizone and cells were maintained in a humidified 5% CO2 incubator at 37°C. Medium was changed every 3 days.

Gene silencing Gene-silencing experiments were performed using siRNA for target genes or nontargeting siRNA as a control (ON-TARGETplus SMARTpool of four siRNAs; Thermo-Scientific, Rochester, NY) according to the manufacturer’s instructions. Briefly, 2 days after plating, the cells were treated with 40 nM siRNA using 5 l/ml RNAiMAX (Invitrogen, Breda, The Netherlands) in antibioticfree complete medium (with FBS). After 6 h of transfection, medium was changed to standard culturing conditions up to day 7.

RNA isolation, cDNA synthesis, and quantitative PCR Total RNA was isolated from HSCs grown in a 24-well plate using RNeasy Mini kit (Qiagen, Venlo, The Netherlands) including the optional on-column DNase digestion (Qiagen RNase-free DNase kit). RNA was dissolved in 30 l of RNase-free water and was quantified spectrophotometrically using a Nanodrop ND1000 (Isogen Life Science, IJsselstein, The Netherlands). An iScript cDNA synthesis kit (Bio-Rad, Veenendaal, The Netherlands) was used to synthesize cDNA. Primer design and quantitative (q) PCR conditions were as described previously (17). Briefly, qPCR reactions were performed in duplicate using the Bio-Rad detection system. Amplifications were carried out in a volume of 25 l containing 12.5 l of 2× SYBR Green Supermix (Bio-Rad), 1 ul of forward and reverse primer, and 1 l cDNA. Cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 45 cycles of denaturation (95°C for 10 s), annealing temperature (see supplementary Tables 1, 2) for 30 s, and elongation (72°C for 30 s). To determine the relative expression of a gene, a 4-fold dilution series from a pool of all samples of all genes tested was used. The amplification efficiency was between 95 and 105%, amplicon sequencing confirmed the specificity and for each sample a melt-curve analysis was performed. IQ5 real-time PCR detection system software (Bio-Rad) was used for data analysis. Expression levels were normalized by using the average relative amount of the reference genes. Reference genes used for normalization are based on their stable expression in stellate cells, namely, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz), hypoxanthine phosphoribosyl transferase (Hprt), and hydroxylmethylbilane synthase (Hmbs). Primers of reference and target genes are listed in supplementary Tables 1, 2.

Immunofluorescence Freshly isolated HSCs were grown on glass coverslips in 24-well plates at 37°C for 7 days. Staining of cells was performed as follows: cells were fixed in 4% (v/v) PF at room temperature for 30 min and stored in 1% (v/v) PF at 4°C for a maximum of 1 week. HSCs were washed twice in PBS, permeabilized [0.1% (w/v) saponin] and blocked with 2% BSA for 1 h at room temperature. After blocking, slides were incubated 1 h with the primary antibody against -SMA (50–75 ug/ml), washed again, and incubated for 1 h with anti-mouse antibody (15 ug/ml) supplemented with Hoechst (4 ug/ml) for nuclear counterstaining and LD dye, LD540 (0.05 ug/ml). Thereafter, coverslips were mounted with FluorSave on microscopic slides. Image acquisition was performed on a Leica TCS SPE-II confocal microscope at the Center of Cellular Imaging, Faculty of Veterinary Medicine, Utrecht University. To quantify LD size and numbers per cell, confocal images of LD540 (LDs) and Hoechst33342 (nuclei) were analyzed with

TAG metabolism in hepatic stellate cells

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CellProfiler v2.1.1. Recognized LDs and nuclei were overlayed on the original image to confirm the identity. The error rate was 2 days) compared with WT cells (half time

ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells.

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid drople...
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